Viral variants and uses therefor

Abstract

Disclosed are viral variants exhibiting reduced sensitivity to particular agents including nucleoside analogues and immunological mediators such as immunoglobulins and immune cells. Also provided are hepatitis B virus (HBV) variants which exhibit a level of replication fitness in the presence of a nucleoside analogue similar to or greater than in the absence of the nucleoside analogue. The present invention also provides methods of treating HBV infection, including a method for identifying a need to change or otherwise alter an existing therapeutic regimen. Also disclosed are methods for monitoring the development in a subject of an increased HBV load in the presence of a nucleoside analogue. The present invention further provides the use of nucleoside analogue-resistant HBV variants which exhibit a similar or increased replication fitness in the presence of the nucleoside analogue compared to in the absence of the nucleoside analogue to screen for medicaments to treat HBV infection.

1 - 35 . (canceled)  
     
     
         36 . A method for detecting a candidate anti-HBV agent which exhibits inhibitory activity to an HBV, said method comprising the use of an HBV variant comprising a mutant DNA polymerase and optionally a mutant surface antigen (listed below in parenthesis) defined by T474N(P120T), M550V (1195M), M550I(W196S), L526M, W499S/W4990 (F145R), or combinations thereof with the proviso that the HBV variant does not contain an M550V or M550I mutation alone and wherein said HBV variant has a level of replication fitness in the presence of a nucleoside analogue similar to or greater than in the absence of said nucleoside analogue.  
     
     
         37 . The method of  claim 36  wherein the HBV variant is generated using a plasmid vector system or a baculovirus vector system.  
     
     
         38 . The method of  claim 36  comprising: 
 generating a genetic construct comprising a replication competent-effective amount of a genome from an HBV comprising a mutant DNA polymerase and optionally a mutant surface antigen (listed below in parenthesis) defined by T474N(P120T), M550V (1195M), M550I (W196S), L526M, W499S/W499Q (F145R), or combinations thereof which mutant is indicative of a variant which exhibits a replication fitness in the presence of a nucleoside analogue similar to or greater than in the absence of said nucleoside analogue with the proviso that the HBV does not contain an M550V or M5501 mutation alone, said genome contained in a plasmid vector and then transfecting cells with said construct;    contacting said cells, before, during and/or after transfection, with the agent to be tested;    culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and    subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent.    
     
     
         39 . The method of  claim 36  comprising: 
 generating a genetic construct comprising a replication competent-effective amount of a genome from an HBV comprising a mutant DNA polymerase and optionally a mutant surface antigen (listed below in parenthesis) defined by T474N(P120T), M550V (1195M), M550I (W196S), L526M, W499S/W499Q (F145R), or combinations thereof which mutant is indicative of a variant which exhibits a replication fitness in the presence of a nucleoside analogue similar to or greater than in the absence of said nucleoside analogue with the proviso that the HBV does not contain an M550V or M5501 mutation alone, said genome contained in or fused to an amount of a baculovirus genome effective to infect cells and then infecting said cells with said construct;    contacting said cells, before, during and/or after infection, with the agent to be tested;    culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and    subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent.    
     
     
         40 . The method of  claim 36  comprising: 
 generating a continuous cell line comprising an infectious copy of a genome of an HBV comprising a mutant DNA polymerase and optionally a mutant surface antigen (listed below in parenthesis) defined by T474N(P120T), M550V (1195M), M550I (W196S), L526M, W499S/W499Q (F145R), or combinations thereof which mutant is indicative of a variant which exhibits a replication fitness in the presence of a nucleoside analogue similar to or greater than in the absence of said nucleoside analogue with the proviso that the HBV does not contain an M550V or M550I mutation alone, said genome being present in a replication competent effective amount such that said infectious HBV genome is stably integrated in said continuous cell line such as but not limited to 2.2.15 or AD;    contacting said cells with the agent to be tested;    culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent; and    subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent.    
     
     
         41 . The method of  claim 36  wherein the effectiveness of a combination of two or more candidate anti-HBV agents is determined.